A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be important to gain a detailed understanding of the impact of malaria on the generation and maintenance of immunological memory. This project represented a collaborative effort between Dr. Pierce and Dr. Louis Miller and his colleagues in the Malaria Vaccine Development Branch (MVDB) and with scientists at the Malaria Research and Training Center (MRTC) at the University of Mali. [unreadable] The hallmark of adaptive immunity is antigen-specific immunological memory. Immunological memory is a phenomenon that having been exposed to a pathogen and survived the infection the experience is remembered by the immune system such that upon re-exposure to the same pathogen an individual's immune response is more rapid and stronger such that the individual may experience no clinical systems of the infection. Indeed, all vaccines are predicated on the phenomenon of immunological memory. However, despite its importance we still have an incomplete understanding of the cellular and molecular mechanisms that underlie the generation, maintenance and activation of immunological memory. Clearly, current efforts to develop a malaria vaccine would benefit from an in-depth understanding of the nature of an effective immune response to the parasite that causes malaria. Over the last year we have focused our efforts on gaining an understanding of the generation, maintenance and activation of B cell memory in response to natural malaria infection.[unreadable] In humans, B cell memory is encoded both in long-lived memory B cells and in plasma cells that reside in the bone marrow. The mechanisms by which memory B cells or plasma cells are generated and maintained over a life time are not known. Current evidence, primarily from serological epidemiological studies, indicates that immunological memory to malaria is slow to be acquired, incomplete and short lived. Thus, despite nearly constant exposure to P. falciparum from birth from infectious mosquito bites, children in endemic areas do not acquire immunity that protects them from severe disease until the age of five. Consequently, children under five years of age are susceptible to severe disease that accounts for over 2 million deaths each year in Africa alone. Acquisition of immunity that protects against severe disease but not against mild disease is acquired prior to adolescence and an immunity sufficient to prevent disease but not to eliminate parasites is acquired only in adolescence to early adulthood. Our current hypothesis is that P. falciparum infection disrupts the normal mechanisms by which B cell memory is generated, maintained or activated. [unreadable] [unreadable] We have taken advantage of recent advances in the identification of antigen-specific human memory B cells in peripheral blood to describe the generation and maintenance of malaria-specific memory B cells in Mali in response to natural infection and vaccination with malaria vaccine candidates currently under development in the MVDB. [unreadable] [unreadable] Over the last year we have continued studies to determine the kinetics of the acquisition of memory B cells with age and the impact of malaria infection on the acquisition and maintenance of memory B cells. To do so we are conducting a longitudinal study on a cohort of 225 volunteers, 2-25 years of age, in a village outside of Bamako, the capital city of Mali. The study was initiated in June 2006 prior to the malaria transmission season which runs July through December. This transmission season with six months of malaria exposure and six months free of malaria offers a near ideal condition to evaluate the impact of malaria infection on B cell memory generation and maintenance. Peripheral blood samples were collected every two months and 14 days after the first case of malaria for each individual. Our field data showed 298 cases of uncomplicated malaria with a predicted gaussian distribution of malaria cases over the transmission season peaking in October. The proportion of individuals who were malaria free decreased in a clear age dependent fashion during the course of the season. A determination of the frequency of hemoglobin types in a multiple linear regression analysis including age and gender showed a surprising finding that an AS hemoglobin type was associated with significant delay to the first malaria infection. This observation will be important to take into account in vaccine trials that assess time to first malaria case as a measure of immunity. Taken together these results indicate that we have acquired quality clinical data on which to interpret the results of our B cell analyses. We followed this cohort through the 2007 malaria seasons collecting peripheral blood samples every two months.[unreadable] [unreadable] Thus far our analysis of this cohort showed that AMA1- and MSP1-specific memory B cells increase with age reflecting a combination maturation of the immune system and continued exposure to malaria. The number of total memory B cells did not change with the transmission season, however, we did observe a decrease in the number of AMA1- and MSP1-specific memory B cells at the peak of the transmission season and 14 days after the first case of malaria that we attributed to a movement of antigen-specific B cells into the lymphoid tissues. The number of memory B cells specific for AMA1 and MSP1 are not significantly different from individuals in the U.S. vaccinated with MSP1 and AMA1 vaccines. However, we found that the quality of the memory B cells are quite different with a large percent of memory B cells showing what has been recently described as an exhausted phenotype. We are currently analyzing these exhausted B cells to determine the molecular basis of the phenotype. Results from studies underway to access the impact of vaccination on these cells should be of interest. [unreadable] [unreadable] We have also initiated studies to assess the nature of the antibody response to P. falciparum in our Kambila cohort in collaboration with Dr. P. Felgner (U.C. Irvine) using a proteome chip containing approximately half of the P. falciparum proteome. Our results thus far indicate that: the intensity and complexity of the antibody response increases with age, that the intensity and complexity of the serum antibodies at the beginning of the malaria season predicts protection from malaria, the complexity and intensity of antibodies transiently rises during the season but most of the increases are lost by six months and do not predict protection. We plan to continue to analyze the data focusing on the top 20 antigens as future vaccine candidates, and to validate the findings in additional sites that differ in the genetic background of the inhabitants and transmission rate. When completed these studies will provide the first comprehensive analysis of the impact of malaria on the generation and maintenance of memory B cells.